IL2RB Is a Prognostic Biomarker Associated with Immune Infiltrates in Pan-Cancer

Background . Interleukin-2 receptor β (IL2RB) is a receptor protein of interleukin-2. IL2RB is implicated in regulation of T cell-mediated immune response. However, the role of IL2RB in pan-cancer is unknown. The present premise sought to explore the role of IL2RB in tumorigenesis, tumor metabolism, and tumor immunity in pan-cancer. Methods . Data were retrieved from multiple data resources including GTEx data resource, CCLE data resource, TCGA data resource, UCSC data resource, and TISIDB web server. These data were adopted to assess the expression, prognosis value, relationship between IL2RB and immune microenvironment, microsatellite instability, immune new antigen, gene mutation, immune modulatory factors immune checkpoint and TMB, and immune or molecular subtypes of IL2RB in various tumors. Estimate analysis and GSEA were conducted to assess the role of IL2RB in pan-cancer. Results . Di ﬀ erential analysis illustrated that L2RB was remarkably elevated in pan-cancer, notably in solid tumors compared with normal tissues. Survival analysis indicated that IL2RB was linked to pan-cancer prognosis, and elevated IL2RB contents were remarkably linked to dismal prognosis patients in diverse kinds of cancers. The ﬁ ndings illustrated that IL2RB contents were remarkably linked to tumor immune invasion, tumor microenvironment, TMB, MSI, DNA repair genes, methyl transferases, immune modulatory factors, and immune or molecular subtypes in pan-cancer. IL2RB gene mutation was evident in numerous cancers. The data illustrated that IL2RB contents were remarkably enriched in multiple signaling cascades which modulate tumorigenesis, tumor metabolism along with immunity. Conclusion . The ﬁ ndings of the present premise illustrate that IL2RB plays an indispensible role in tumorigenesis, tumor metabolism, and immunity. Therefore, it is a prospective target gene in tumor-target therapy and tumor immune therapy. IL2RB is also a valuable predictive biomarker in most solid tumors.


Introduction
According to the most recent report from the International Agency for Research on Cancer (IARC), about 19.3 million new cases of cancer along with 10.0 million cancer-linked deaths were recorded globally in 2020 [1]. Despite significant progress in diagnosis and treatment of malignant tumor in recent years, most cancer types present poor prognosis. This is mainly attributed to lack of effective diagnostic methods and treatment strategies. Hence, it is pivotal to explore novel biomarkers for diagnosis along with prognosis of cancers to improve cancer management.
Interleukin-2 (IL-2) is a primary immunoregulatory cytokine produced from activated T cells. IL-2 exerts its effects by combining with IL-2R (interleukin-2 receptor) expressed on T cells along with natural killer (NK) cells [2,3]. IL-2R comprises three subunits: IL-2Rα (coded by IL2RA), IL-2Rβ (coded by IL2RB), and IL-2Rγ (coded by IL2RG) [4,5]. IL2RB gene is a cytokine signaling gene that participates in T celltriggered immune responses. Studies report that IL2RB is 2.2. Expression Analysis of IL2RB in Pan-Cancer. Differences in IL2RB contents between tumor tissues and nontumorous tissues were evaluated using R software. Difference in the contents of IL2RB between different nontumorous cells and diverse cancer cell lines were explored using Kruskal-Wallis test. Violin plots were constructed via the ggplot R package.

Prognostic Analysis of IL2RB in Pan-Cancer.
Univariate survival assessment was performed to explore the association of IL2RB contents with survival of patients. Besides, the Kaplan-Meier approach was utilized to explore the survival rate in pan-cancer with different contents of IL2RB. Tumor along with vicinal non-tumorous tissue samples were categorized into high-IL2RB expression and low-IL2RB expression groups on the basis of the expression levels of IL2RB using the bipartite approach. Univariate Cox survival assessment was performed using survival R package. Data were visualized forest plots.

Association
Analysis of IL2RB with the Immune Microenvironment. Tumor-invading lymphocytes are a reliable predictor of sentinel lymph node status as well as cancer survival. Cancer patients' survival is linked to their immune along with stromal scores. Our research method was similar to the former study [9]; the ESTIMATE tool was adopted to explore the relationship of gene expression with immune cell scores. A positive relationship was defined by P < :05 coupled with R > 0:20.

Association
Analysis of IL2RB with Immune Neoantigens along with Immune Checkpoints Genes. A mutated gene in malignant cells codes for neoantigen. The protein is produced as a result of biological events, consisting of point mutations and gene fusions, as well as deletion mutations. The docking affinity score of neoantigen was calculated via antigenic epitopes harboring 8~11 amino acids long. Epitopes harboring a score of less than 500 nm were considered as neoantigens. The predicted neoantigens were ranked according to the docking affinity and variant allele frequency along with antigenicity index values. Using the ScanNeo tool, as similar as research method of former study [9], the number of neoantigens in each tumor sample was measured independently. The link between IL2RB contents with antigen number was also investigated. Moreover, the expression relationship between the top 40 immune checkpoint genes and IL2RB expression was explored. The immune checkpoint genes were abstracted separately, and the correlation between their expression levels and IL2RB expression was determined. P < 0:05 and R > 0:20 indicated remarkably positive correlations.

Association Analysis of IL2RB with Tumor Mutational
Burden and Microsatellite Instability. Tumor mutational burden (TMB) is a measurable marker for quantifying mutations present in a tumor cell. We employed Spearman's rank correlation to assess the TMB of each cancer sample. When comparing tumor tissue to nonmalignant tissue, microsatellite instability (MSI) involves the presence of a novel microsatellite allele. It constitutes any change in the length of a microsatellite produced by the insertion or deletion of a repeat unit. We utilized the Spearman's rank correlation to explore the relationship of IL2RB contents with MSI.

2
Journal of Oncology  3 Journal of Oncology 2.7. Association Analysis of IL2RB with DNA Mismatch Repair Genes and Methyltransferases. Mismatch repair is a process for repairing mismatches inside cells. When critical genes lose their function as a result of this process, DNA replication mistakes occur that cannot be corrected. These mistakes result in a significant number of somatic mutations. Using expression profile data from the TCGA data resource, the association of five MMR genes (MSH6, MLH1, EPCAM, MSH2, and PMS2) with IL2RB contents was investigated. DNA methylation is a chemical alteration of DNA that affects epigenetic inheritance and modulates gene expression without changing the DNA sequence. The link between IL2RB contents and the expression of four methyltransferases was explored. The ggplot R tool was utilized to visualize the data. The associations were remarkably positive at P < 0:05 along with R > 0:20.

Gene Set Enrichment Analysis of IL2RB in Pan-Cancer.
Gene set enrichment analysis (GSEA) compares genes to predetermined gene sets in order to assess their expression status within a functional gene set. It is also utilized to assess if the expression level is linked to a biological process, a molecular function, or a cellular component [10]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) constitute a comprehensive data resource for genomic, chemical, as well as functional analyses. The Hallmark gene set was employed in GSEA analysis via the molecular signatures database (MsigDB) [11]. The following criteria were used in the GSEA analysis: |NES| >1, P < :05, and FDR 0.25. Cascades that satisfied these criteria were deemed to be remarkably enriched [9]. Expression levels of IL2RB in a dataset containing 21 tissues in tumor cell lines, collected from the CCLE. (c) Expression levels of ATG5 in tumor and paired adjacent noncancerous tissues containing 20 tissues from TCGA, * P < 0:05, * * P < 0:01, and * * * P < 0:001. (d) IL2RB expression difference in 27 tumors integrating data of normal tissues in GTEx database and data of TCGA tumor tissues, * P < 0:05, * * P < 0:01, and * * * P < 0:001.  7 Journal of Oncology records in 2530 studies. The web server is used for analysis of target genes in the tumor immune interplay via analyses of high-throughput data or literature abstraction [12]. Herein, TISIDB was utilized to generate heat maps for exploring the relationship between the contents of IL2RB with immunomodulators and immune cells in diverse kinds of cancer. Further, the relationship of IL2RB contents with immune subtypes or molecular subtypes across human cancers was explored using TISIDB. Besides, the relationship of target genes of some antitumor drugs and il2rb gene was evaluated using TISIDB web server. Statistical significance was defined at P < :05.

Results
3.1. IL2RB Is Upregulated in Pan-Cancer. The contents of IL2RB in numerous tissues were determined using GTEx data resources, and the expression trend of IL2RB in 31 tissues was described (Figure 1(a)). The expression levels of IL2RB in 21 cell lines were evaluated using data for tumor cell lines retrieved from CCLE data resource (Figure 1(b)). The mRNA contents of IL2RB were abstracted from 20 cancers in TCGA dataset, and a box diagram of the contents of IL2RB in cancers tissues and neighboring tissues was generated ( Figure 1(c)). The contents of IL2RB transcript were

IL2RB Is a Potential Prognosis Marker in Pan-Cancer.
The relationships of the IL2RB mRNA contents with OS (overall survival), DSS (disease-specific survival), DFS (disease-free survival), and PFI (progression-free interval) of 44 cancers were explored. Kaplan-Meier (KM) survival curve coupled with Cox proportional hazards models were utilized to evaluate the prognosis value of IL2RB. The results for Cox regression analyses and correlations between IL2RB with OS, DSS, DFI, and PFI in various cancers were presented as forest plots. The data illustrated that elevated IL2RB contents were not remarkably correlated with DFI ( Figure 2 The results of the KM curve analysis showed that high IL2RB mRNA contents were linked to worse OS in GBM, KIRP, LGG, THYM, and UVM, whereas high IL2RB mRNA levels were associated with better OS in HNSC, SKCM, and SARC ( Figure 3(a)). In addition, highIL2RB mRNA levels were correlated with worse DFI in KIRP; worse DSS in GBM, KIRP, LGG, and UVM; and worse PFI in DLBC, KIRP, and LGG. High IL2RB mRNA levels were correlated with better DFI in UCEC, better DSS in SKCM and UCEC, and better PFI in CHOL and UCEC (Figures 3(b)-3(d)).

IL2RB Expression Is Linked to Immune Neoantigens and
Immune Checkpoint Genes. To explore the relationship between IL2RB contents with checkpoint gene expression, we combined data from more than forty immune checkpoint genes often detected in diverse kinds of malignancies ( Figure 6). The data illustrated that the contents of IL2RB were positively linked with the contents of immune checkpoint genes consisting of THCA, PRAD, and COAD in diverse kinds of malignancies. We established that L2RB modulates the expression of numerous immune checkpoint genes, which plays a pivotal role in modifying tumor immunity. To assess the relationship of IL2RB contents with the amount of these neoantigens, the number of neoantigens in each kind of tumor was evaluated independently.

12
Journal of Oncology genomic CpG dinucleotides by DNA methyltransferases. We visually explored the association of IL2RB contents with the expression of four methyltransferases and established that IL2RB contents were positively linked to the vast number of cancers, as illustrated in Figure 11. These data illustrated that IL2RB may modulate tumorigenesis along with progression in human pan-cancer via modulating epigenetic status.

IL2RB Modulates Signaling Cascades Involved in Cancer
Metabolism along with Tumor Immunity. To assess the influence of gene expression on cancers, human pancancer samples were stratified into two groups on the basis of the IL2RB contents: high and low. We utilized GSEA to determine the signaling cascades enriched in the KEGG and HALLMARK data resources in both high and low expression groups. Tables 1 and 2 exhibit the top 20 most enriched signaling cascades or biological processes on the basis of the NES score permutation. We selected the top 3 signaling cascades most enriched in the data resources (Figures 12(a)-12(b)). Cytokine-receptor interaction and complement were the most enriched signaling pathways. These data illustrated that IL2RB plays an indispensable role in modulating signaling cascades participating in tumor immunity along with metabolism.

Relationship between IL2RB and Immune Modulatory
Factors in Pan-Cancers. The relationship between IL2RB expression and abundance of tumor-infiltrating lymphocytes (TILs) was explored to determine the role of IL2RB in immune system and its implication in progression of cancers. The data illustrated that IL2RB contents were linked to relative abundance of TILs (such as Act CD8, Tem CD8, and BTLA  CD200  TNFRSF14  NRP1  LAIR1  TNFSF4  CD244  LAG3  ICOS  CD40LG  CTLA4  CD48  CD28  CD200R1  HAVCR2  ADORA2A  CD276  KIR3DL1  CD80  PDCD1  LGALS9  CD160  TNFSF14  IDO2  ICOSLG  TMIGD2  VTCN1  IDO1  PDCD1LG2  HHLA2  TNFSF18  BTNL2  CD70  TNFSF9  TNFRSF8  CD27  TNFRSF25  VSIR  TNFRSF4  CD40  TNFRSF18  TNFSF15  TIGIT  CD274  CD86  CD44  TNFRSF9  ACC  BLCA  BRCA  CESC  CHOL  COAD  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  LUAD  LUSC  MESO  OV  PAAD  PCPG  PRAD  PEAD  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC   15 Journal of Oncology Figure 11: Correlation analysis between the expression of IL2RB in pan-cancer and the expression levels of four types of methyltransferases. DNMT1 is colored red, DNMT2 was colored blue, DNMT3a was colored green, and DNMT3b is colored purple. Pearson's rho -Log10 (p value) Figure 10: Correlation analysis between the expression of IL2RB in pan-cancer and the expression levels of DNA repair genes. 16 Journal of Oncology Th1) in most human cancers ( Figure 13). Associations between IL2RB expression with immunoinhibitors, immunostimulators, MHCs, chemokines, and receptors were inferred using gene set variation analysis on the basis of the gene expression profile. IL2RB expression levels were positively linked to multiple levels of immunoinhibitors (such as CD96, PDCD1, and TIGIT), immunostimulators (such as CD27, CD28, and CD86), most MHCs (such as   ex ex x ex x x x x _ _ _ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Hig Hig Hig Hig ig Hig Hig H Hig Hig Hig Hig i Hig Hig Hig Hig g Hig ig Hig H Hig Hi ig Hig Hig g ig Hig g Hig Hig g Hig g H g g g g g g g Hig Hig H g g g g g g g g g g g g g g g gh e h_e    Journal of Oncology 3.9. IL2RB Expression Is Associated with Immune and Molecular Subtypes in Pan-Cancers. The TISIDB website was adopted to examine the impact of IL2RB contents on immunological, as well as molecular subtypes in Pancancers. C1 (wound healing), C4 (lymphocyte deficient), C2 (IFN-gamma dominant), C5 (immunologically quiet), C3 (inflammatory), and C6 (TGF-b dominant) are the six kinds of immune subtypes. The data exhibiting differential IL2RB expression was among the immune subtypes in various cancers. Expression level of IL2RB was remarkably different among immune subtypes in each cancer type. The top 4 cancer types with the most significant differential IL2RB expression are presented in Figure 14. Further, different molecular subtypes of cancers were remarkably associated with expression of IL2RB.The top 4 tumor types with the most significant differential expression of IL2RB are presented in Figure 15. The results indicate that IL2RB expression differs in immune subtypes and molecular subtypes of diverse human cancers.

h_e h h_e h h h_ h h e h h h_e h h h_e h h h e h h h_ h_ h_e h h_e h h_ h h h h h_e h h h h h_e h_ h h h h_
3.10. IL2RB Is a Target Gene of Several Antitumor Drugs. Data were retrieved from Drug Bank to explore whether IL2RB is a potential target for antitumor drugs. The findings showed that IL2RB gene was a potential target gene for antitumor drugs such as Denileukin diftitox, Aldesleukin, Daclizumab, and Basiliximab ( Figure 16 and Table 3). This indicated that IL2RB is a valuable target for antitumor drugs. However, further studies should be conducted to explore the effect of targeting this protein on tumor progression.

Discussion
Interleukin-2 (IL-2) along with the IL-2 receptor (IL-2R) is the key factor in the regulation of immune system homeostasis and tolerance. IL-2 constitutes a T cell growth factor, which promotes proliferation along with differentiation of activated T cells. IL2RB is part of a receptor signaling complex with pleiotropic functions [13]. IL2RB is activated by endogenous IL2 or therapeutic stimulation, resulting in expression of antitumor immune cell cytokines, such as CD8+ T cells, NK cells, and CD4 + T cells. Previous investigations report that IL2RB is remarkably associated with lung cancer, colorectal cancer, and some autoimmunity diseases [14][15][16]. However, the role of IL2RB in Pan-cancers has not been fully elucidated.
The findings of the present work exhibited higher contents of IL2RB in pan-cancers, especially in solid tumors in contrast with nontumorous tissues. Moreover, high expres-sion of IL2RB was correlated with worse prognosis in the most tumor types. This indicates that IL2RB plays a key role in tumorigenesis, tumor proliferation, and tumor metastasis.
Tumor invading lymphocytes in TME are independent predictors for cancer prognosis and immunotherapeutic efficacy [17,18]. Studies report that tumor immune microenvironment is linked to tumorigenesis along with tumor progression [19,20]. The data herein exhibited that the IL2RB contents were positively linked to the abundance of tumor infiltrating lymphocytes, for instance, CD8 + T cells, dendritic cells, and macrophages in pan-cancers. The expression of IL2RB in ACC, BRCA, and CHOL was positively linked with the abundance of macrophages, which are implicated in immune evasion and suppression [21]. Moreover, more than 40 types of immune-related checkpoint genes including CD274 (PD-L1) were used to further explore the immune-related role of IL2RB. PD-L1 inversely modulates T cell proliferation along with cytokine secretion

25
Journal of Oncology at a given threshold of T cell receptor attack by binding to PD-1. In addition, it maintains the peripheral tolerance and impairs the immune function of T cell in TME [22,23]. The findings in the present work indicated that IL2RB expression was remarkably linked to PD-L1 function in most cancer types. Notably, IL2RB was co-expressed with PDCD1 gene, which encodes PD-1 protein. LAG3 is a negative regulator of T cells, and previous findings indicate that blocking LAG3 improves proliferation and activity of cytotoxic T lymphocyte. LAG3 is co-expressed with PD-1 in tumor invading lymphocytes in cancer tissues. Targeting LAG3 combined with PD-1 blockade can alleviate tumor progression and increase regression [24,25]. The data herein illustrated that IL2RB contents were remarkably linked to LAG3 expression in various cancers. A similar expression trend for IL2RB, PD-1L, and LAG3 in tumors imply that these proteins promote tumor aggressiveness through a common cascade; however, further studies should verify these findings. TMB and MSI remarkably modulate tumor phenotype and patient outcomes [26,27]. In this premise, IL2RB expression was remarkably linked to TMB or MSI in diverse cancers. However, the effect of IL2RB on TMB or MSI was different in different cancer types. Several investigations are currently exploring tumor immunotherapy to evaluate recovering of antitumor immunoreaction by rebooting and maintaining tumor immune circulation. In

26
Journal of Oncology the present premise, IL2RB expression was remarkably linked to the expression of key target genes for cancer immunotherapy in pan-cancer. These genes include immunoinhibitors such as CD96 [28,29], immunostimulators such as CD27 [30], MHCs such as B2M [31], chemokines such as CXCL9 [32], and receptors such as CCR2 [33,34]. These data illustrate that IL2RB is a potential target gene for development of tumor immunotherapy. IL2RB is receptor of IL-2, which regulates human immunity by binding IL-2. Studies report that mutations in IL2RB gene reduce surface expression and IL-2 binding, causing T and NK cell-driven immune dysregulation. In addition, high expression of wild-type IL2RB increases IL-2 responsiveness of human T lymphocytes in vitro [35,36]. Similarly, the results in the current study showed mutation of IL2RB gene in numerous kinds of cancers, implying IL2RB gene plays the role of an oncogene in some cancer types.
Gene set enrichment analysis was performed to explore the role of IL2RB in pan-cancer that was performed for high as well as low IL2RB expression groups. The data illustrated that IL2RB is implicated in several signaling pathways, such as compliment signaling, cytokine-cytokine receptor interaction signaling, PI3K-Akt-mTOR signaling, and allograftrejection signaling. The highest enrichment scores of these signaling cascades were observed in the high-expression sub-group of IL2RB.This indicates that high IL2RB expression positively modulates these signaling pathways. Moreover, these data illustrate that IL2RB plays a vital role in promoting immune activation, modulating tumor cell viability, and promoting proliferation of tumor cell by upregulating the related signaling cascades.

Conclusion
In summary, IL2RB expression level is higher in pan-caner tissues relative to the expression level in normal tissues. High IL2RB expression level is associated with worse clinical prognosis in pan-cancers. TMB, MSI, MMRs, and DNA methylation dysregulate IL2RB expression in pan-cancer. In addition, IL2RB is remarkably linked to tumor immune microenvironment and is a potential therapeutic target gene for tumor immunotherapy.

Data Availability
The underlying data supporting the results of our study can be found in GTEx data resource, CCLE data resource, TCGA data resource, UCSC data resource and TISIDB webserver.